A growing body of information suggests that abnormal intracellular Ca2+ regulation plays a role in neurodegeneration. For example, there is evidence that depleting Ca2+ within the endoplasmic reticulum can lead to apoptosis, and mitochondrial Ca2+ overload is thought to play a role in excitotoxic cell death. The proposed study will use mouse cerebellar Purkinje neurons as a model to investigate changes in intracellular Ca2+ handling that occur as a result of a mutation (leaner) in the gene encoding the pore-forming subunit of P/Q type voltage-sensitive Ca2+ channels, the dominant voltage-gated Ca2+ channel in these cells. Animals expressing the leaner mutation display profound behavioral symptoms and severe Purkinje cell neurodegeneration. Previous work indicates that Purkinje neurons from leaner mice show modifications in intracellular Ca2+ handling that would be expected to cause redistributions of Ca2+ during stimulation that promote cell death, but the basis for these modifications, and their contribution to neurodegeneration, are unknown. We have developed a conceptual framework and experimental tools in amphibian sympathetic neurons that permit in situ analysis of neuronal Ca2+ transport systems and how they work together as a system in intact cells. These approaches will be applied to the study of intracellular Ca2+ regulation in normal Purkinje neurons, and the Ca2+ transport modifications that are responsible for altered Ca2+ handling in the neurons from leaner mice, with a particular focus on Ca2+ transport modifications that would be expected to increase susceptibility to cell death and contribute to neurodegeneration.